In vivo targeting of ERG potassium channels in mice and dogs by a positron-emitting analogue of fluoroclofilium

The antiarrhythmic clofilium is an efficient blocker of hERG1 potassium channels that are strongly expressed in the heart. Therefore, derivatives of clofilium that emit positrons might be useful tools for monitoring hERG1 channels in vivo. Fluoro- clofilium (F-clofilium) was synthesized and its channel-blocking properties were determined for hERG1 and hEAG1 channels expressed in HEK?293 cells and in Xenopus oocytes. When applied extracellularly in the whole-cell patch-clamp configuration, F-cloflium exhibited a slower onset of block when compared with clofilium, presumably owing to its lower membrane permeability. When applied in the inside-out configuration at the intracellular membrane side, it blocked hEAG1 channels almost as efficiently as clofilium (IC50 1.37 nM and 0.83 nM, respectively). Similar results were obtained for hERG1, showing F-clofilium is a potent hERG1 and hEAG1 channel blocker once it has reached the intracellularly accessible target site at the channel. Using the (18)F-labeled analog we studied the in vivo binding and distribution of F-clofilium in mice and a dog. Greatest activity was found in kidneys and bones. A small but significant enrichment of activity in the dog myocardium known for its expression of cERG1 channels allowed to depict the myocardium of a living dog by PET. Thus, F-clofilium is a useful tool for imaging hERG channels in living organisms.     

A multiscale simulation system for the prediction of drug-induced cardiotoxicity

The preclinical assessment of drug-induced ventricular arrhythmia, a major concern for regulators, is typically based on experimental or computational models focused on the potassium channel hERG (human ether-a-go-go-related gene, K(v)11.1). Even if the role of this ion channel in the ventricular repolarization is of critical importance, the complexity of the events involved make the cardiac safety assessment based only on hERG has a high risk of producing either false positive or negative results. We introduce a multiscale simulation system aiming to produce a better cardiotoxicity assessment. At the molecular scale, the proposed system uses a combination of docking simulations on two potassium channels, hERG and KCNQ1, plus three-dimensional quantitative structure-activity relationship modeling for predicting how the tested compound will block the potassium currents IK(r) and IK(s). The obtained results have been introduced in electrophysiological models of the cardiomyocytes and the ventricular tissue, allowing the direct prediction of the drug effects on electrocardiogram simulations. The usefulness of the whole method is illustrated by predicting the cardiotoxic effect of several compounds, including some examples in which classic hERG-based models produce false positive or negative results, yielding correct predictions for all of them. These results can be considered a proof of concept, suggesting that multiscale prediction systems can be suitable for being used for preliminary screening in lead discovery, before the compound is physically available, or in early preclinical development when they can be fed with experimentally obtained data.     

Methods of screening,monitoring and management of cardiac toxicity induced by chemotherapeutics

Cardiac toxicity is one of the most common side effects of anticancer drugs. Cardiac toxicity results dysfunction of heart including hypotension, heart failure, and even cause death in extreme cases. The potential risk of cardiotoxicity is a huge concern in chemotherapeutics mediated cancer treatment. The individual with any pre-existing cardiac issues are excluded from clinical trials due to the potential risk of cardiotoxicity. Because of the potential cardiotoxicity, there is an emerging need...     

Combined receptor and ligand-based approach to the universal pharmacophore model development for studies of drug blockade to the hERG1 pore domain

Long QT syndrome, LQTS, results in serious cardiovascular disorders, such as tachyarrhythmia and sudden cardiac death. A promiscuous binding of different drugs to the intracavitary binding site in the pore domain (PD) of human ether-a-go-go related gene (hERG) channels leads to a similar dysfunction, known as a drug-induced LQTS. Therefore, an assessment of the blocking ability for potent drugs is of great pragmatic value for molecular pharmacology and medicinal chemistry of hERGs. Thus, we attempted to create an in silico model aimed at blinded drug screening for their blocking ability to the hERG1 PD. Two distinct approaches to the drug blockage, ligand-based QSAR and receptor-based molecular docking methods, are combined for development of a universal pharmacophore model, which provides rapid assessment of drug blocking ability to the hERG1 channel. The best 3D-QSAR model (AAADR.7) from PHASE modeling was selected from a pool consisting of 44 initial candidates. The constructed model using 31 hERG blockers was validated with 9 test set compounds. The resulting model correctly predicted the pIC(50) values of test set compounds as true unknowns. To further evaluate the pharmacophore model, 14 hERG blockers with diverse hERG blocking potencies were selected from literature and they were used as additional external blind test sets. The resulting average deviation between in vitro and predicted pIC(50) values of external test set blockers is found as 0.29 suggesting that the model is able to accuretely predict the pIC(50) values as true unknowns. These pharmacophore models were merged with a previously developed atomistic receptor model for the hERG1 PD and exhibited a high consistency between ligand-based and receptor-based models. Therefore, the developed 3D-QSAR model provides a predictive tool for profiling candidate compounds before their synthesis. This model also indicated the key functional groups determining a high-affinity blockade of the hERG1 channel. To cross-validate consistency between the constructed hERG1 pore domain and the pharmacophore models, we performed docking studies using the homology model of hERG1. To understand how polar or nonpolar moieties of inhibitors stimulate channel inhibition, critical amino acid replacement (i.e., T623, S624, S649, Y652 and F656) at the hERG cavity was examined by in silico mutagenesis. The average docking score differences between wild type and mutated hERG channels was found to have the following order: F656A > Y652A > S624A > T623A > S649A. These results are in agreement with experimental data.     

A risk assessment of human ether-a-go-go-related gene potassium channel inhibition by using lipophilicity and basicity for drug discovery

The blockade of human ether-a-go-go-related gene (hERG) potassium channels is widely regarded as the predominant cause of drug-induced QT prolongation. The correlation analysis between the inhibition of the hERG channel (hERG inhibition) and physicochemical properties was investigated by use of in-house quinolone antibiotics as model compounds. In order to establish a simple prediction model of hERG inhibition, we focused on the comprehensible physicochemical parameters such as lipophilicity (log P) and basicity (pK(a)). At first, the risk associated with increasing log P and pK(a) was examined by statistical analysis. It was demonstrated that the risk associated with increasing log P and pK(a) by one unit, respectively, almost identically increased. Consequently, equal attention should be paid to both parameters on hERG inhibition. Next, a prediction model of hERG inhibition which was represented by log P and pK(a) was investigated. As a result, we built the stepwise discriminant prediction model which took advantage of the risk judgment by zone classification. In conclusion, the impact of log P and pK(a) on hERG inhibition was clarified relatively and quantitatively. The quantitative risk assessment established based on both parameters, was considered to be a practical and useful tool in avoiding hERG inhibition and in the rational drug design for drug discovery, especially in lead optimization. Moreover, we also carried out a trend analysis using a different derivative and demonstrated that both parameters were equally significant for hERG inhibition.     

In silico predictions of hERG channel blockers in drug discovery: from ligand-based and target-based approaches to systems chemical biology

The risk for cardiotoxic side effects represents a major problem in clinical studies of drug candidates and regulatory agencies have explicitly recommended that all new drug candidates should be tested for blockage of the human Ether-a-go-go Related-Gene (hERG) potassium channel. Indeed, several drugs with different therapeutic indications and recognized as hERG blockers were recently withdrawn due to the risk of QT prolongation, arrhythmia and Torsade de Pointes. In silico techniques can provide a priori knowledge of hERG blockers, thus reducing the costs associated with screening assays. Significant progress has been made in structure-based and ligand-based drug design and a number of models have been developed to predict hERG blockage. Although approaches such as homology modeling in combination with docking and molecular dynamics bring us closer to understand the drug-channel interactions whereas QSAR and classification models provide a faster assessment and detection of hERG-related drug toxicity, limitation on the applicability domain of the current models and integration of data from diverse in vitro approaches are still issues to challenge. Therefore, this review will emphasize on current methods to predict hERG blockers and the need of consistent data to improve the quality and performance of the in silico models. Finally, integration of network-based analysis on drugs inducing potentially long-QT syndrome and arrhythmia will be discussed as a new perspective for a better understanding of the drug responses in systems chemical biology.     

Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.     

A critical assessment of combined ligand- and structure-based approaches to HERG channel blocker modeling

Blockade of human ether-à-go-go related gene (hERG) channel prolongs the duration of the cardiac action potential and is a common reason for drug failure in preclinical safety trials. Therefore, it is of great importance to develop robust in silico tools to predict potential hERG blockers in the early stages of drug discovery and development. Herein we described comprehensive approaches to assess the discrimination of hERG-active and -inactive compounds by combining quantitative structure-activity relationship (QSAR) modeling, pharmacophore analysis, and molecular docking. Our consensus models demonstrated high-predictive capacity and improved enrichment and could correctly classify 91.8% of 147 hERG blockers from 351 inactives. To further enhance our modeling effort, hERG homology models were constructed, and molecular docking studies were conducted, resulting in high correlations (R2 = 0.81) between predicted and experimental pIC??s. We expect our unique models can be applied to efficient screening for hERG blockades, and our extensive understanding of the hERG-inhibitor interactions will facilitate the rational design of drugs devoid of hERG channel activity and hence with reduced cardiac toxicities.     

A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels

Cancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4‐nitrobenzenesulfonyl (DNS) protected 2‐hydroxyisophthalamide system that exploits GSH for its activation into free 2‐hydroxyisophthalamide forming supramolecular M⁺/Cl^? channels. Better permeation of the DNS protected compound into MCF‐7 cells compared to the free 2‐hydroxyisophthalamide and GSH‐activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH‐activatable transport‐mediated cell death was further validated in rat insulinoma cells (INS‐1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF‐7 cells with efficiency similar to that of the anticancer drug doxorubicin.     

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

Structure‐based drug development is often hampered by the lack of in vivo activity of promising compounds screened in vitro, due to low membrane permeability or poor intracellular binding selectivity. Herein, we show that ligand screening can be performed in living human cells by “intracellular protein‐observed” NMR spectroscopy, without requiring enzymatic activity measurements or other cellular assays. Quantitative binding information is obtained by fast, inexpensive ¹H NMR experiments, providing intracellular dose‐ and time‐dependent ligand binding curves, from which kinetic and thermodynamic parameters linked to cell permeability and binding affinity and selectivity are obtained. The approach was applied to carbonic anhydrase and, in principle, can be extended to any NMR‐observable intracellular target. The results obtained are directly related to the potency of candidate drugs, that is, the required dose. The application of this approach at an early stage of the drug design pipeline could greatly increase the low success rate of modern drug development.     

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.     

On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network

This paper proposes a new cytotoxicity assay in a microfluidic device with microwells and a distributive microfluidic channel network for the formation of cancer cell spheroids. The assay can generate rapid and uniform cell clusters in microwells and test in situ cytotoxicity of anticancer drugs including sequential drug treatments, long term culture of spheroids and cell viability assays. Inlet ports are connected to the microwells by a hydraulic resistance network. This uniform distribution of cell suspensions results in regular spheroid dimensions. Injected cancer cells were trapped in microwells, and aggregated into tumor spheroids within 3 days. A cytotoxicity test of the spheroids in microwells was subsequently processed in the same device without the extraction of cells. The in situ cytotoxicity assay of tumor spheroids in microwells was comparable with the MTT assay on hanging drop spheroids using a conventional 96-well plate. It was observed that the inhibition rate of the spheroids was less than that in the 2D culture dish and the effect on tumor spheroids was different depending on the anticancer drug. This device could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids, offering more information than the conventional 2D culture plate.     

Design,Synthesis, and In Vitro Evaluation of Novel Indolyl DiHydropyrazole Derivatives as Potential Anticancer Agents

Indoles derived from both natural sources or artificial synthetic methods have been known to interact with aryl hydrocarbon receptors (AhR), and exhibit anticancer activity. In light of these attractive properties, a series of hybrid molecules with structural features of indoles, i.e., those bearing a pyrazoline nucleus, were evaluated for their enhanced anticancer activity. The designed molecules were subjected to molecular docking in order to screen for potential AhR interacting compounds, and the identified indolyl dihydropyrazole derivatives were synthesized. The synthesized compounds were characterized, and their cytotoxicity was evaluated against four human cancer cell lines using the MTT assay. Based on the Glide g-score, H-bonding interactions and bonding energy of 20 candidate molecules were selected for further analysis from the 64 initially designed molecules. These candidate molecules have shown promising anti-proliferative activity against the cell lines tested. Among these candidate molecules, the compounds with hydroxy phenyl substitution on the pyrazoline ring have shown potent activity across all the tested cell lines. The designed scaffold was proven effective for screening potential candidate molecules with anticancer properties, and may be further optimized structurally for yielding the ideal anti-tumorigenic compound for the treatment of various cancers.     

A short-term NaCl exposure increases the Na(+) conductance of outward-rectified cation currents in the pith cells of sweet pepper

The regulatory role of pith cells in the stem in Na(+) recirculation in sweet pepper was investigated by evaluating the transport characteristics of the plasma membrane of this cell type and comparison with those of root cells. Ion conductivity and Na(+) permeability of the plasma membranes of protoplasts of both cell types were studied with the patch-clamp technique in the whole-cell configuration, before and after addition of NaCl to the bath medium. Protoplasts of both pith and root cells showed outward rectifying currents with a reversal potential (V(r)) near to the equilibrium potential of K(+) (EK). Addition of NaCl to the bath medium caused a stronger shift of the reversal potential, V(r), in pith protoplasts than in root protoplasts, indicating that the outward rectified currents are permeable to Na(+), especially in the pith cells.After plant exposure to exogenous NaCl via the nutrient solution for 1 week, V(r) in the root cells was closer to EK than in the control plants and hardly shifted upon addition of Na(+). This indicated that the net permeability of the OR channel complement in the plasma membrane to Na(+) was lower following exposure to Na(+). V(r) in the pith protoplasts, on the other hand, shifted significantly more than in the control plants, suggesting an increase of the permeability to Na(+). Moreover, the Na(+) channel blocker amiloride blocked the currents in this cell type. It is concluded that pith cells have appropriate features of outward rectified currents to enable Na(+) accumulation or release when NaCl is present in or removed from the nutrient medium. Probably, exogenous NaCl even induced expression and formation of Na(+)-permeable channels in pith cells.     

Synthesis,Physical Properties,and Cytotoxicity of Nitroxyl–Aziridine Hybrid

Nitroxyl–aziridine hybrid, a candidate for a magnetic resonance imaging (MRI) probe and an anticancer drug, was synthesized by aziridine formation reaction of nitroxyl‐introduced aldehyde and guanidinium ylide in good diastereoselectivity. The relative configuration at aziridine C(2) C(3) bond of the major diastereoisomer was determined to be cis by X‐ray crystallographic analysis. Application of chiral guanidinium ylide resulted in the formation of the corresponding optically active aziridine in 84% ee. Reversible one‐electron redox potential and quantitative spin yield of the hybrid were observed in cyclic voltammogram and electron spin resonance, respectively. However, cytotoxicity of the hybrid against cancer cell lines used was not observed.